U.S. patent number 5,994,498 [Application Number 08/915,827] was granted by the patent office on 1999-11-30 for method of forming water-soluble, electrically conductive and optically active polymers.
This patent grant is currently assigned to Massachusetts Lowell, University of Lowell. Invention is credited to K. Shridhara Alva, Jayant Kumar, Kenneth A. Marx, Lynne A. Samuelson, Sukant Tripathy.
United States Patent |
5,994,498 |
Tripathy , et al. |
November 30, 1999 |
**Please see images for:
( Certificate of Correction ) ** |
Method of forming water-soluble, electrically conductive and
optically active polymers
Abstract
Water-soluble polymers are formed by combining a water-soluble
analog of a water-insoluble monomer, such as a water-insoluble
redox monomer, with a water-based solvent and an enzyme. The
water-soluble polymers formed can be electrically conductive or
optically active. The water-soluble analog can be copolymerized
with a water-insoluble redox monomer to form a copolymer that is
also water-soluble. Polymers formed by the method of this invention
can be layered on a surface to form, for example, alternating
layers of polyanions and polycations.
Inventors: |
Tripathy; Sukant (Acton,
MA), Samuelson; Lynne A. (Marlboro, MA), Alva; K.
Shridhara (Lowell, MA), Kumar; Jayant (Westford, MA),
Marx; Kenneth A. (Francestown, NH) |
Assignee: |
Massachusetts Lowell, University of
Lowell (MA)
|
Family
ID: |
25436313 |
Appl.
No.: |
08/915,827 |
Filed: |
August 21, 1997 |
Current U.S.
Class: |
528/422; 528/487;
528/491; 528/499 |
Current CPC
Class: |
H01B
1/128 (20130101); C08G 73/0266 (20130101) |
Current International
Class: |
C08G
73/00 (20060101); C08G 73/02 (20060101); H01B
1/12 (20060101); C08G 073/00 () |
Field of
Search: |
;528/422,487,491,499 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Truong; Duc
Attorney, Agent or Firm: Hamilton, Brook, Smith &
Reynolds, P.C.
Government Interests
GOVERNMENT SUPPORT
This invention was made with support from the Government under ARO
URI Grant DAAL03-91-G-0064, ARO Cooperative Grant DAAH04-94-2-003
and US Army/USDA Contract DAAK60-93-K-0004. The Government has
certain rights in this invention.
Claims
We claim:
1. A method of forming a water-soluble polymer, comprising the step
of combining a redox monomer having at least one charged
substituent with a water-based solvent and an enzyme, to form a
reaction mixture that causes the redox monomer to polymerize,
thereby forming the water-soluble polymer.
2. The method of claim 1, wherein the reaction mixture further
includes a second redox monomer, wherein said second redox monomer
is water-insoluble whereby the water-soluble polymer formed is a
copolymer.
3. The method of claim 1, wherein the redox monomer is a
substituted aniline.
4. The method of claim 3, wherein the redox monomer is
2,5'diaminobenzene sulfonate.
5. The method of claim 3, wherein the redox monomer is 4,4'diamino
stilbene-2,2'disulfonic acid.
6. The method of claim 3, wherein the redox monomer is
o-aminobenzene sulfonic acid.
7. The method of claim 3, wherein the redox monomer is
p-aminobenzene sulfonic acid.
8. The method of claim 3, wherein the redox monomer is
p-aminobenzoic acid.
9. The method of claim 3, wherein the redox monomer is sulfanilic
acid.
10. The method of claim 1, wherein the redox monomer is a
substituted phenol.
11. The method of claim 10, wherein the redox monomer is
tyrosine.
12. The method of claim 10, wherein the redox monomer is
p-hydrobenzoic acid.
13. The method of claim 10, wherein the redox monomer is phenol
red.
14. The method of claim 10, wherein the redox monomer is
dopamine.
15. The method of claim 10, wherein the redox monomer is acid
red.
16. The method of claim 1, wherein the substituent is a cation.
17. The method of claim 1, wherein the substituent is an anion.
18. The method of claim 3, wherein the substituent is at an ortho
position.
19. The method of claim 3, wherein the substituent is at the para
position.
20. The method of claim 1, wherein the enzyme is a peroxidase.
21. The method of claim 20, wherein the peroxidase is horseradish
peroxidase.
22. The method of claim 20 further including the step of combining
hydrogen peroxide with the reaction solution.
23. The method of claim 1, wherein the redox monomer is a dye.
24. The method of claim 23 wherein the redox monomer is an azo
compound.
25. The method of claim 1, wherein the redox monomer is a
ligand.
26. The method of claim 1, wherein the reaction mixture has a pH in
a range of greater than about 4.
27. The method of claim 26, wherein the reaction mixture has a pH
of between about 6 and about 8.
28. The method of claim 1, wherein the substituent is a sulfonate
group.
29. The method of claim 1, wherein the substituent is a carboxyl
group.
30. The method of claim 1, wherein a first substituent is a cation
and a second substituent is an anion.
31. The method of claim 25, wherein the redox monomer is a
substituted hydroxyquinoline.
32. The method of claim 1, wherein the water-soluble polymer is
electrically conductive.
33. The method of claim 31, wherein the water-soluble polymer has
an electrical conductivity of about 10.sup.-8 S/cm to 10.sup.-1
S/cm.
34. The method of claim 1, wherein the water-soluble polymer is
optically active.
35. The method of claim 10, wherein the substituent is at an ortho
position.
36. The method of claim 10, wherein the substituent is at the para
position.
37. The method of claim 1, wherein the reaction mixture further
includes an unsubstituted redox monomer, whereby the water-soluble
polymer formed is a copolymer.
Description
BACKGROUND OF THE INVENTION
Electrically conductive and optically active polymers have been
known for many years. Examples of electrically conductive polymers
include polythiophene, polypyrrole and polyaniline. Recently, there
has been an increased interest in development of such polymers for
application to a wide range of uses, such as, for example,
light-weight energy storage devices, electrolytic capacitors,
anti-static and anti-corrosive coatings for smart windows and
biological sensors. However, the application of electrically
conductive and optically active polymers has been limited by some
fundamental properties of monomers employed to form these polymers
and by processing limitations that limit the quality of the
resulting polymers.
Among the most limiting problems of electrically conductive and
optically active polymers is their lack of water solubility.
Typically, therefore, these polymers are formed in an organic
solvent. Attempts to increase the water solubility of these
polymers have included derivatization of the polymer following its
formation. However, derivatization of electrically conductive and
optically active polymers requires several steps and generally
proceeds under relatively harsh reaction conditions including, for
example, use of fuming sulfuric acid. Further, such derivatization
typically results in only partial substitution and, therefore, the
improvement in water solubility is limited. In addition, polymers
typically degrade during the derivatization, thereby further
limiting the effectiveness of post-reaction attempts to improve
water solubility.
Another attempt to improve the water solubility of electrically
conductive and optically active polymers has been derivatization of
monomers and subsequent polymerization in an organic solvent.
However, the polymerization rate of derivatized monomers is
typically diminished.
Most recently, enzymes, and most notably, horseradish peroxidase,
have been employed to accelerate the reaction rate of derivatized
monomers. Nevertheless, such reactions, in the context of an
organic solvent, generally must be conducted at a relatively low
pH. Further, generation of water molecules as a consequence of
enzyme catalyzed reactions has typically required that such
reactions be conducted in the context of a two-phase reaction
system of aqueous micelles. Use of micelles generally limits the
polymerization of the polymer and presents additional problems with
processing the polymer product, due to the two-phase nature of the
reaction system.
Therefore, a need exists to overcome or minimize the
above-referenced problems associated with formation of
water-soluble electrically conductive and optically active
polymers.
SUMMARY OF THE INVENTION
The present invention relates to a method of forming water-soluble,
electrically conductive and optically active polymers.
In one embodiment, the method includes combining a water-soluble
analog of a water-insoluble redox monomer with a water-based
solvent and an enzyme. A reaction mixture is thereby formed that
causes the analog to polymerize, thereby forming the water-soluble
polymer.
This invention has many advantages. For example, the polymerization
occurs in a water-based solvent, thereby eliminating processing
problems associated with removal of the polymer product from an
organic solvent, or from a reaction system that employs aqueous
micelles. Further, the reaction can be conducted at a higher pH
that is more environmentally neutral and at which the enzyme is
typically more active. Also, the resulting polymers generally are
water-soluble regardless of their molecular weight. Water-soluble
monomers can also be combined with water-insoluble monomers for
polymerization by the method of the invention to form water-soluble
copolymers. The polymers can also be doped with ions present in a
buffer component of the aqueous solvent, thereby causing the
polymer composition to be self-doped.
Polymers formed by the method of the invention can be formed in
higher purity and with greater uniformity, less branching and fewer
co-products or impurities, because the polymer does not need to be
separated from an organic solvent, and because the polymer product
typically does not require subsequent derivatization to obtain the
necessary water-solubility. The polymer product generally is also
more stable than products derivatized after polymerization.
Consequently, the polymers formed by the method of the invention
exhibit greater electrical conductivity and/or optical activity.
Further, polymers formed by the method of the invention are better
suited to many applications, such as self-assembled mono and
multi-layered fabrication of thin film devices and structures.
Also, the polymer products exhibit a greater availability of
functional groups for further molecular engineering, such as
incorporation of biological molecules and of polymers employed to
form biosensors.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows FT-IR spectra of (a) p-aminobenzoic acid monomer and
(b) poly(p-aminobenzoic acid) in KCl formed by the method of the
invention.
FIG. 2 shows absorption spectra of poly(p-aminobenzoic acid) in (a)
acid solution and (b) alkaline solution. The inset gives the
absorption spectra recorded at two minute time intervals during
undoping of the polymer at pH 12.0.
FIG. 3 shows excitation and emission spectra of poly(p-aminobenzoic
acid) in acidic and alkaline solutions. Curves (a) and (b) are the
excitation and emission spectra of the polymer in acidic solution.
Curves (c) and (d) are the excitation and emission spectra in
alkaline solution.
FIG. 4 shows cyclic voltammogram of (a) p-aminobenzoic acid and (b)
poly(p-aminobenzoic acid) in 0.1 M KCl/10 mM ammonium
hydroxide.
FIG. 5 shows change in the absorption spectrum as a function of
time during the polymerization of 2,4 diaminobenzene sulfonate in
Tris-HCl buffer, pH 8.0. The inset shows the change in absorbance
at 420 nm recorded as a function of time during the
polymerization.
FIG. 6 shows proton magnetic resonance spectra during the
polymerization of 2,4 diaminobenzene sulfonic acid in D.sub.2 O:
(a) 2,4 diaminobenzene sulfonic acid with horseradish peroxidase;
(b) four minutes after addition of hydrogen peroxide; (c) nine
minutes after addition of hydrogen peroxide; and (d) eighteen
minutes after addition of hydrogen peroxide.
FIG. 7 shows FT-IR spectra of (a) diaminobenzene sulfonate and (b)
poly(diaminobenzene sulfonate) in KCl.
FIG. 8 shows absorption spectra of poly(2,4 diaminobenzene sulfonic
acid) in solutions of different pH values. (a) 1.2; (b) 3.0; (c)
6.0; (d) 9.0; (e) 10.0; and (f) 12.8.
FIG. 9 shows plot of change in absorbance as a function of
concentration recorded at pH 10.0 for the polymer. The inset gives
the optical density at 470 nm as a function of concentration
measured at a pH of 6.0.
FIG. 10 shows excitation and emission spectra of
poly(diaminobenzene sulfonate) at: pH 1.2 (plots a and d); pH 3.0
(plots b and e); and pH 6.0 (plots c and f). The intensities in
plots c and f have been divided by two in this representation. The
spectra above pH 6.0 are identical to those at pH 6.0.
FIG. 11 shows cyclic voltammograms of (a) 2,4 diaminobenzene
sulfonic acid and (b) poly(2,4 diaminobenzene sulfonic acid) under
nitrogen atmosphere. The potential at the platinum wire working
electrode was varied at a scan rate of 50 mV/s with respect to
Ag/AgCl reference electrode. A platinum mesh electrode was used as
counter electrode.
FIG. 12 shows absorption spectrum of 10 bilayers of poly 2,5
diaminobenzene sulfonate (PDABSA) and poly(diallyl dimethyl
ammonium chloride) (PDDAC) deposited by a layer-by-layer technique
of the invention. The inset shows the absorption maximum at 535 nm
as a function of bilayers.
FIG. 13 shows absorption spectra of phenylazoaniline and
poly(phenylazoaniline) in dimethyl formamide (DMF).
FIG. 14 shows relaxation of absorption spectra of
poly(phenylazoaniline) in DMF after exciting with an argon ion
laser for ten minutes.
FIG. 15 shows absorption spectra of (a) diaminoazobenzene and (b)
poly(diaminoazobenzene) in DMF.
FIG. 16 shows relaxation of absorption spectra of
poly(diaminoazobenzene) in DMF after exciting with an argon ion
laser for ten minutes.
FIG. 17 shows C-13 spectra of 8-hydroxy quinoline-5-sulfonate (HQS)
and poly(8-hydroxy quinoline-5-sulfonate) (PHQS) in D.sub.2 O
recorded using a Bruker.RTM. 200 MHz NMR spectrometer.
FIG. 18 shows a plot of absorbance as a function of concentration
of the ligand for a fixed concentration of FeCl.sub.3, measured at
440 nm.
FIG. 19 shows a change in absorbance as a function of FeCl.sub.3
concentration for a fixed concentration of the polymeric
ligand.
DETAILED DESCRIPTION OF THE INVENTION
The features and other details of the apparatus and method of the
invention will now be more particularly described and pointed out
in the claims. It will be understood that the particular
embodiments of the invention are shown by way of illustration and
not as limitations of the invention. The principal features of this
invention can be employed in various embodiments without departing
from the scope of the invention. All parts and percentages are by
weight unless otherwise specified.
The method of the invention includes forming a water-soluble
polymer by polymerizing a water-soluble analog of a water-insoluble
monomer. Preferably the water-insoluble monomer is a redox monomer.
A "redox monomer," as defined herein, is a monomer that will
polymerize by a redox reaction. The polymerization reaction occurs
in a reaction mixture that includes, in addition to the
water-soluble analog of the water-insoluble redox monomer, a
water-based solvent and an enzyme.
The reaction solution is formed by adjusting the pH of a suitable
water-based solvent. Preferably, the solvent is water. However,
other components of the solvent can include, for example, dimethyl
formamide, methanol, ethanol, dioxane, etc. The pH of the
water-based solvent is adjusted to a pH in a range of between about
5.0 and about 8.0. In one embodiment, the pH can be adjusted to a
range of between about 6.0 and about 7.0. Preferably, the pH is
about 6.0. Examples of suitable buffers include Tris-HCl buffer,
sodium phosphate, etc. Preferably, the buffer is Tris-HCl
buffer.
A suitable enzyme is added to the reaction mixture. The
concentration of enzyme in the reaction mixture is sufficient to
significantly increase the polymerization rate of the monomer in
the reaction solution. Typically, the concentration of enzyme in
the reaction mixture is in a range of between about one unit/ml and
about five units/ml, where one unit will form 1.0 mg purpurogallin
from pyrogallol in 20 seconds at pH 6.0 at 20.degree. C.
Examples of suitable enzymes include peroxidases, laccase, etc.
Preferred enzymes are peroxidases. A particularly preferred enzyme
is horseradish peroxidase.
A water-soluble analog of a water-insoluble redox monomer is added
to the reaction mixture. The concentration of water-soluble analog
generally in the reaction mixture is in a range of between about 10
mm and about 100 mm. The water-soluble monomer is suitable for
enzyme-catalyzed polymerization to form a water-soluble
polymer.
Applicants have discovered that water-soluble analogs for
water-insoluble redox monomers can be polymerized to form
water-soluble polymers. Examples of suitable water-soluble analogs
include water-soluble analogs of anilines, phenols, etc. In one
embodiment, the water-soluble analogs include analogs of anilines
that also include azo groups, whereby the resulting polymers are
optically active.
The water-soluble analogs of the water-insoluble redox monomer
generally include a substituent at the ortho- or para-position of
an aromatic redox monomer. The substituents can carry a negative or
a positive charge when in an aqueous-based solvent. Examples of
suitable substituents include carboxyl, sulfonic, phosphonic
groups, etc.
The polymerization reaction is initiated by adding a suitable
oxidant, such as a hydrogen peroxide solution, etc. In one
embodiment, the hydrogen peroxide has a concentration in the
solution in a range of between about one millimolar (mm) and about
five millomolars. Preferably, the concentration of hydrogen
peroxide in the solution added to the reaction mixture is about
30%. The reaction mixture is stirred while adding the hydrogen
peroxide solution. Typically, the reaction mixture is maintained at
a temperature in a range of between about 10.degree. C. and about
35.degree. C. during polymerization. Preferably, the temperature of
the reaction mixture is maintained at a temperature of about
20.degree. C. during polymerization.
It is believed that polymerization of a water-soluble analog of a
water-insoluble redox monomer in a water-based solvent that
includes an enzyme component enables regular polymerization of the
analog to form linear polymers by free-radical polymerization, and
that the resulting polymers will remain water-soluble, often
regardless of their molecular weight.
In addition to water-soluble analogs of water-insoluble redox
monomers, the reaction mixture can include water-insoluble redox
monomers. Copolymerization of the water-insoluble analogs and
water-insoluble redox monomers generally will result in
water-soluble copolymers. In one embodiment, the molar ratio of
water-soluble analogs and water-insoluble redox monomers is in a
range of between about 1:9 and about 9:1. It is to be understood
that copolymerization can include more than one water-soluble
analog, or a water-soluble analog of one water-insoluble redox
monomer and a second water-insoluble redox monomer, or more. The
resulting polymer can be a self-doped polymerization in the
presence of Tris-HCl buffer. The polymer can be undoped
subsequently by raising the pH of the polymer solution.
In another embodiment, the method of the invention includes
polymerization of the water-soluble analog on a surface, whereby a
layer of the polymer is formed. In this embodiment, the pH of the
polymer solution is reduced to a suitable pH, such as a pH in a
range of between about 2.0 and about 8.0, by adding a suitable
acid, such as hydrochloric acid, etc. A suitable surface, such as a
glass slide treated with an alkali, such as CHEMSOLV.RTM., is
immersed in a polymer solution for a sufficient period of time to
cause the polymer to accumulate at the surface. In one embodiment,
a glass slide is immersed in a polymer solution for about ten
minutes and then removed. The surface can then be washed with water
at a pH of about 2.5 in order to remove unbound polymer from the
surface.
Distinct layers of polymers can be applied to a surface by this
method. For example, an initial layer can be formed by exposing a
suitable surface to a polymer formed by the method of the invention
that is a polycation and then subsequently exposing the same
surface, having the polycation deposited upon it, into a solution
of a polyanion formed by the method of the invention. In one
specific embodiment, a glass slide treated with CHEMSOLV.RTM. is
exposed to a one milligram/milliliter solution of poly(diallyl
dimethyl ammonium chloride) at a pH of 2.5 as a polycation, and
then exposed to a one milligram/milliliter solution of two poly(2,5
diaminobenzene sulfonate) formed by the method of the invention, as
a polyanion. A bilayer of polymers is thereby formed. Additional
layers of these or other polymers can subsequently be applied.
It is to be understood that polymers formed by the method of the
invention can be formed ranging from an oxidized, electrically
conducting form to a reduced, insulating form of the polymer. It is
also to be understood that the polymers formed by the method of the
invention can be modified after polymerization. For example,
modification can be made at amine functional groups to form amides
or imine groups.
Dissolved polymers formed by the method of the invention can be
precipitated from solution by reducing the pH with a suitable acid.
Examples of suitable acids include hydrochloric acid, etc.
The invention will now be further and more specifically described
by the following examples. All parts and percentages are by weight
unless otherwise specified.
EXAMPLE 1
Materials and Methods
Horseradish peroxidase (HRP) (enzyme classification number (EC)
1.11.1.7) and Tris-HCl buffer were obtained from Sigma Chemicals
Company, St. Louis, Mo. p-Aminobenzoic acid (ABA) and hydrogen
peroxide were obtained from Aldrich Chemicals, Inc., Milwaukee,
Wis. All the chemicals were used as received.
The enzymatic polymerization of ABA was achieved by HRP catalyzed
oxidative free radical coupling. 300 mg of ABA was dissolved in 20
mL of 0.1M Tris-HCl buffer, pH 6.0 containing 300 units of HRP. The
reaction was initiated with 200 .mu.L of 30% hydrogen peroxide with
continuous stirring. The reaction was allowed to continue for three
hours. The pH of the reaction medium was lowered to 1.0 where the
polymer precipitates. The precipitate was filtered off to obtain a
dark powder of PABA.
Perkin-Elmer LAMBDA-9.RTM. UV-Vis-near IR spectrophotometer was
used for the spectral characterization of the polymer. The
fluorescence experiments were carried out using a SLM 8100
spectrofluorometer. The structural characterization of the polymer
was carried out by using NMR and FT-IR spectroscopic techniques.
.sup.1 H NMR spectra were recorded in D.sub.2 O, using a Bruker 250
MHz NMR Spectrometer. FT-IR experiments were carried out using a
Perkin-Elmer FT-IR spectrophotometer. Electrochemical properties of
the polymer and the monomer were studied in a three electrode setup
(EG&G Applied Princeton Research Potentiostat/Galvanostat model
263, Princeton, N.J.) consisting of a platinum wire working
electrode. A platinum mesh electrode and a silver/silver chloride
electrode were used as counter and reference electrodes
respectively. All experiments were carried out under nitrogen
atmosphere with prior saturation of the electrolyte with
nitrogen.
RESULTS AND DISCUSSION
Enzyme catalyzed polymerization of ABA proceeded rapidly. The
change in absorption spectrum was used to monitor the progress of
the polymerization reaction. Absorption spectra changed very
rapidly in the first couple of minutes. Subsequently, the change in
absorbance was small and became negligible after about 15 min.
However, in the bulk polymerization reactions, the reaction was
allowed to continue for at least three hours before precipitating
the polymer.
The polymer was characterized by FT-IR and NMR spectroscopic
techniques. FIG. 1 is a plot of the FT-IR spectrum of the polymer
compared to that of the monomer. It was observed from the figure
that the NH stretching in the polymer had a broad band at 3450
cm.sup.-1 which appeared as two clear peaks at 3450 and 3350
cm.sup.-1 in the case of the monomer. The IR bands in lower energy
regions of the spectrum broadened upon polymerization, with the
disappearance of peaks at frequencies such as 1700, 1650 and 1450
cm.sup.-1. The polymerization was also confirmed by proton NMR
spectroscopy. The monomer gave two doublets at 7.83 ppm and 6.88
ppm corresponding to the aromatic protons. Upon polymerization,
these peaks shifted to 7.82 ppm and 6.89 ppm, respectively, along
with the appearance of three broad peaks at 8.04, 7.21 and 7.05
ppm. The appearance of multiple peaks suggested that both types of
bonds, as shown in a proposed reaction mechanism, below, were
present in the resulting polymer. The average molecular weight of
the polymer was about 3000 daltons. The proposed reaction mechanism
is as follows: ##STR1##
The polymer was doped with the ions present in the buffer. The self
doping of the polymer was established by recording the absorption
spectrum of the polymer at various pH conditions. FIG. 2 shows two
typical absorption spectra of the polymer covering the acidic and
alkaline regimes. The absorption characteristics of the polymer did
not show any drastic change up to a pH of 10.0. Above pH 10.01 the
solution color changed to green which subsequently turned to yellow
after about 15 min. The inset in FIG. 2 shows a set of absorption
spectra recorded with a time interval of two minutes during
undoping at a pH of 12.0. This suggests that the undoping of PABA
followed a slow reaction kinetics. The emission characteristics of
PABA also differ in its doped and undoped forms, as shown in FIG.
3. The excitation and emission spectra of doped PABA (a and b) are
broader as compared to that of the undoped PABA (curves c and d).
The doped form of the polymer had an emission maximum at 400 nm
(curve b) while that of the undoped form shifted to 360 nm (curve
d). Curves (a) and (b) are the excitation and emission spectra of
the polymer in acidic solution. Curves (c) and (d) are the
excitation and emission spectra in alkaline solution. The
conductivity of the as-synthesized self-doped polymer was in the
semiconducting regime (10.sup.-5 Siemens/cm).
The electrochemical activity of PABA was established by recording a
cyclic voltammogram of the polymer. FIG. 4 shows the cyclic
voltammograms of the monomer and the polymer recorded at 100 mV/s.
The monomer underwent reversible redox reaction, with a redox
potential of -0.55 V with respect to an Ag/AgCl electrode. Upon
polymerization, the reduction potential shifted to -0.2 V with a
large charging current in the reverse cycle. The plot of the peak
current verses the square root of the scan rate followed the
Randles-Sevcik relationship (peak current proportional to the
square root of the scan rate), indicating that the polymer and the
monomer were electrochemically reversible redox systems.
In conclusion, a self-doped water-soluble polyaniline was
synthesized from p-aminobenzoic acid by a biochemical method. The
polymer was polyionic and could be used for the development of self
assembled mono and multilayers for the fabrication of thin-film
devices and structures (Ferreira, M., et al., "Thin solid films,"
244:806 (1994)). The polymer had functional groups available for
further molecular engineering, such as incorporation of biological
molecules for biosensor applications (Alva, K. S. et al.,
"Proceedings of SPIE: Smart Materials Technologies and
Biomimetics," 2716:152 (1996)).
EXAMPLE 2
Materials
Horseradish peroxidase and Tris-HCl buffer were obtained from Sigma
Chemicals Co, St. Louis, Mo. 2,4 Diaminobenzene sulfonic acid
(DABSA) was obtained from Aldrich Chemicals Company, Inc.,
Milwaukee, Wis. All other chemicals and solvents used were of
analytical grade or better and used as obtained.
The infrared spectrum was recorded with a Perkin-Elmer 1760X FTIR
spectrometer. The UV-Vis spectra were recorded using a Perkin-Elmer
LAMBDA-9.RTM. UV/VIS/NIR spectrophotometer. The emission
characteristics of the polymer were studied using a SLM 8100
spectrofluorometer. The electrochemical characterization of the
polymer was carried out using a Potentiostat (EG&G
Potentiostat/Galvanostat Model 263). A platinum wire was used as
the working electrode. The potential was applied with respect to a
silver/silver chloride electrode using platinum mesh as the counter
electrode. The reaction was carried out in 0.1M Tris-HCl buffer, pH
6.0, under nitrogen atmosphere. The molecular weight was determined
using Gel Permeation Chromatography utilizing Waters Model 510 pump
and Waters Model 410 refractive index detector with Jordi columns
relative to polystyrene standards. Dimethyl formamide (DMF)
containing 1% LiBr was used as the eluent.
Enzymatic Synthesis of the Polymer
The polymerization of DABSA was carried out in 0.1M Tris-HCl
buffer, pH 6.0. 0.1 g of DABSA was dissolved in 50 ml of Tris
buffer containing 3 units of the enzyme. The reaction was initiated
with the addition of 100 .mu.l of 30% hydrogen peroxide with
stirring. The polymerization reaction started instantaneously. The
reaction was allowed to continue at room temperature for a minimum
of 3 hours with constant stirring. The reaction medium was dialyzed
against water to remove the buffer. The polymer was then extracted
with methanol, which was later evaporated off to obtain dark brown
colored polymer with 80% yield.
Thin Films by Layer-By-Layer Technique
Self assembly of the polyaniline poly(DABSA) on a glass slide was
carried out by the layer-by-layer deposition technique (Ferreira,
M., et al., "Thin solid films," 244:806 (1994)). A glass slide
treated with alkali (CHEMSOLV.RTM.) was exposed to polycation and
polyanion solutions repeatedly to transfer monolayers of these
polyelectrolytes per every exposure. 1 mg/ml solution of
poly(diallyl dimethyl ammonium chloride) (PDDAC) at pH 2.5 was used
as the polycation while 1 mg/ml solution of PDABSA also at pH 2.5
was used as the polyanion. The glass slide was exposed to the
polyelectrolyte solution for 10 minutes and washed with water at pH
2.5 to remove the unbound polymer from the surface. This process
was repeated to obtain the desired number of bilayers.
RESULTS AND DISCUSSION
KINETIC EXPERIMENTS
The polymerization reaction was followed by UV-Vis spectroscopy. In
this experiment, the concentration of hydrogen peroxide, DABSA and
the solution pH were chosen such that the reaction rate was low
enough to be followed by UV-Vis spectroscopy. FIG. 5 shows a
typical set of absorption spectra of DABSA (1 mg/100 ml) recorded
during the polymerization in Tris-HCl buffer at pH 8.0, with one
minute time intervals after the initiation of the polymerization
with 10l of 3% hydrogen peroxide. The inset in FIG. 5 shows the
change in absorbance recorded at 420 nm, corresponding to the
absorption maximum of the polymer, as a function of time. The
changes in absorbance were dramatic in the initial stages of the
reaction, which attained a steady state in about 15 minutes. We
also observed that the maximum conversion of DABSA is achieved at a
pH of 6.0. Therefore, the bulk polymerization was carried out at pH
of 6.0. In the bulk polymerization, the reaction was allowed to
continue for 3-4 hours with intermittent addition of hydrogen
peroxide to ensure completion of the reaction. The reaction medium
was then dialyzed against water and extracted with ethanol. GPC
analysis showed that the polymer has a molecular weight (M.sub.w)
of the order of 18000 daltons.
NUCLEAR MAGNETIC RESONANCE STUDIES
The polymerization process was also followed by in-situ NMR
spectroscopy. The polymerization reaction was carried out in
D.sub.2 O (sodium phosphate buffer pH 6.0) in a NMR tube. Reaction
was initiated by the addition of 2 .mu.l of 30% hydrogen peroxide
and the NMR spectra were recorded at different reaction time
intervals. Characteristic spectra recorded during the
polymerization are given in FIG. 6. FIG. 6(a) is the spectrum of
the monomer containing the enzyme, before the addition of hydrogen
peroxide. It can be observed from FIG. 6(a) that the monomer showed
one singlet and a doublet corresponding to the aromatic protons.
The spectral widths are very broad due to the low solubility of the
monomer. The molar concentration of the enzyme was very low
compared to DABSA, hence its protons did not appear in the NMR
spectrum. FIG. 6(b), 6(c) and 6(d) represent the spectra recorded 4
minutes, 9 minutes and 18 minutes after the addition of hydrogen
peroxide. As the polymerization reaction progressed, the peak
pattern changed, with the appearance of new peaks. These peaks
arose from the change in the chemical environment of the aromatic
protons upon oxidative free radical coupling. The other striking
observation was that the peaks became sharper with the progress of
the polymerization reaction. This could be ascribed to the reduced
intermolecular hydrogen bonding due to the disappearance of free
amine groups upon polymerization, which resulted in improved
solubility of the polymer as compared to the monomer. The time
scale of the completion of the polymerization reaction is in
agreement with the observed 420 nm saturation in the UV-Visible
spectroscopic studies.
FT-IR SPECTROSCOPY
The FT-IR spectra DABSA and its polymer in KCl matrix are shown in
FIG. 7. The monomer (curve a) shows characteristic amine vibration
bands at 3430 cm.sup.-1 with a shoulder at 3200 cm.sup.-1. The
polymer (curve b) on the other hand shows a broad peak centered
around 3450 cm.sup.-1 with a shoulder around 2940 cm.sup.-1. The
peaks at lower energy regions also become broader upon
polymerization. The number of vibrational bands in the ring
hydrogen rocking regime (1250-1000 cm.sup.-1) is lower in the case
of polymer as compared to the monomer, indicating the disappearance
of the ring hydrogens upon polymerization.
ABSORPTION CHARACTERISTICS
FIG. 8 shows the effect of pH on the absorption spectrum. The stock
solution of the polymer at pH 7.0 was diluted to a constant
dilution in 0.1M KCl solution at various final pH values. It can be
observed from the figure that the absorption characteristics
underwent a series of changes upon increasing the solution pH from
1.3 to 12.8. The absorption at 540 nm decreased with increasing pH
while a new peak appeared around 445 nm upon increasing the
solution pH. The absorption band at 540 nm was assigned to the
doped form of the polymer while at 445 nm the conjugated polymer
exhibited its characteristic absorption band. The conversion from
the doped to the undoped form of the polymer was instantaneous, as
observed by spectral changes, indicating that the undoping kinetics
was rapid. The polymer could be shuttled between its doped and
undoped forms by the proper choice of solution pH. FIG. 9 shows the
absorption spectra of the polymer recorded as a function of
concentration at pH 1.2. The absorbance increases linearly with
concentration. The inset presents the absorbance at the peak
maximum (540 nm) measured at a pH 6.0 plotted as a function of the
concentration of the polymer. The absorbance follows a linear
relationship with concentration.
EMISSION CHARACTERISTICS
FIG. 10 shows the excitation and emission spectra of the polymer at
various pH conditions. The emission characteristics show an
interesting pH dependence. The polymer at pH 1.2 had emission
(curve d) only in the blue region. The emission maximum was 380 nm
with an excitation maximum at 320 nm (curve a). Upon increasing the
pH to 3.0, a new emission band at 530 nm (curve e) appeared with an
intensity comparable to that at 380 nm. The excitation spectrum
(curve b) shows multiple bands with peak maxima at 340, 380 and 460
nm unlike at pH 1.2. Upon increasing the pH to 6.0, the emission at
530 nm (curve f) increased and the intensity was about two orders
of magnitude higher than that of low pH solution. The excitation
spectrum was identical to that at pH 3.0 except for the intensity.
These observations indicate that the polymer existed predominantly
in the acid form at pH 1.2. At pH 3.0, the doped and undoped forms
of the polymer coexisted. At pH 6.0, the undoped form of the
polymer dominated in the solution. Similar spectral features were
observed at pH values above 6.0. This dependence of the
characteristic emission features on solution pH could be attributed
to the pKa values of the amine groups (.about.3.0).
ELECTROCHEMICAL PROPERTIES
FIG. 11 examines the cyclic voltammograms of the monomer and the
polymer at a platinum electrode at pH of 6.0. Curve (a) is the
cyclic voltammogram of the monomer in the presence of horseradish
peroxidase. The peak current followed a linear relation with the
square root of the scan rate indicating that the monomer was a
component of an electrochemical redox system. The monomer was
allowed to polymerize in the reaction cell by the addition of 10
.mu.l of 30% hydrogen peroxide and the reaction was allowed to
continue for 30 minutes. Curve (b) is the cyclic voltammogram of
the reaction mixture after 30 minutes of enzymatic polymerization.
Upon polymerization, the peak currents at monomer redox potentials
have decreased, with the appearance of a new reduction potential at
-0.225V corresponding to the polyaniline. The oxidation potential
(+0.52V with respect to Ag/AgCl) of the polymer was identical to
that of the monomer. However, the oxidizable species were lower in
concentration in the case of polymer resulting in a lower peak
current. The peak current followed a linear relationship with the
square root of the scan rate, as in the case of the monomer,
indicating that the polymerization was also a reversible redox
system.
The polymer, as synthesized, showed a conductivity in the
semiconducting regime (10.sup.-5 S/cm). The low conductivity could
be attributed to the complex structure of the polymer. The
conductivity of the polymer could be improved by co-polymerizing
with underivatized aniline with a stoichiometry such that
solubility was still maintained.
THIN FILMS BY LAYER-BY-LAYER TECHNIQUE
HISTORY OF LAYER-BY-LAYER TECHNIQUE
The solubility of the polyaniline described in this study at all pH
conditions made it a perfect candidate for the fabrication of thin
films by the layer-by-layer technique. This polymer can be used as
polyanion with another polycation of interest. The preliminary
studies on fabrication of multilayers by this technique indicated
that multilayers of this polyaniline can be prepared at any pH
condition with a proper choice of the polycation. FIG. 12 shows the
absorption spectrum of ten bilayers of polyaniline with PDDAC
deposited on a glass slide at pH 2.5. Since PDDAC was not absorbing
in the spectral region scanned in this study, the absorbance was
solely due to the polyaniline. The absorption spectrum of the
multilayer assembly shows the absorption maxima at 535 nm and 450
nm, respectively. The inset in FIG. 12 shows the absorbance
recorded at 535 nm as each bilayer was deposited on the glass
slide. The constant change in absorbance per bilayer indicates that
thin films can be built with precise control over thickness and
organization. Similar observation was also made at neutral pH
conditions.
EXAMPLE 3
MATERIALS AND METHODS
Horseradish peroxidase was obtained from Sigma Chemicals Co., St.
Louis, Mo. All aniline and phenol derivatives were purchased from
Aldrich Chemicals Co. Milwaukee, Wis. in the purest form possible.
All the chemicals were used as obtained.
Enzyme-catalyzed polymerization of phenols and anilines were
carried out in aqueous media unless otherwise mentioned. The
monomer solution containing the enzyme in a Tris-HCl buffer
solution at a pH of 6.5 was treated with hydrogen peroxide under
ambient conditions. The reaction was allowed to continue for about
three hours. The water-soluble polymers were dialyzed against water
and the resulting polymer was extracted to DMF. In the case of
water-insoluble polymers, the polymers were filtered off and washed
with water to remove the unreacted monomer and the biocatalyst.
All spectroscopic characterization of the polymer and
polymerization reaction were done using a Perkin-Elmer
LAMBDA-9.RTM. UV-Vis-near IR spectrophotometer. The vibration
spectra of the polymers were recorded using Perkin-Elmer FT-IR
spectrophotometer. The proton and C-13 NMR spectra were recorded on
a Bruker 200 MHz NMR spectrometer. The electrochemical properties
of the polymers were studied using a Potentiostat (EG&G
potentiostat/Galvanostat model 263) in a three electrode setup
containing platinum wire, Ag/AgCl and platinum mesh as the working,
reference and counter electrode in nitrogen saturated Tris-HCl
buffer at pH 6.5.
RESULTS AND DISCUSSION
POLY(P-AMINO BENZOIC ACID)
Poly(p-amino benzoic acid) was obtained by HRP catalyzed
polymerization of p-amino benzoic acid in Tris-HCl buffer at pH 6.0
(Alva, K. S., et al., "Macromolecular Rapid Communications" 17:000
(1996)). The polymer was precipitated as dark brown solid by
lowering the solution pH. The precipitate was filtered off and
washed with acidified water to remove unreacted monomer and the
enzyme. The polymer was soluble in water under neutral and alkaline
conditions. The polymer had a molecular weight on the order of 3K
daltons. The polymer was characterized by FT-IR spectroscopy. The
peak positions at 3450cm-1 and the disappearance of the ring
rocking frequencies were indicative of the polymerization. The
optical properties of the polymer are influenced by the solution pH
conditions. A plot of absorption and emission properties indicated
that absorbance at longer wavelength disappeared upon increasing
the solution pH. It has been observed that the spectral features
remained unchanged until a pH value of 10 was reached, and
thereafter the absorbance at 520 nm decreased. It has also been
observed that the decrease in the absorbance at 520 nm was
indicative of a slow reaction. These changes in absorbance upon
increasing the solution pH were ascribed to undoping of the
polymer. A cyclic voltammogram of the polymer in 0.1M KCl solution
the presence of 10 mM ammonium hydroxide indicated that a peak
current followed a linear relationship with the square root of the
scan rate, indicating that the polymerization was a reversible
redox system. The polymer displayed a large charging current at
positive potential during the oxidation cycle. The polymer as
synthesized showed a conductivity on the order of 10.sup.-5
S/cm.
POLY(2,5 DIAMINOBENZENE SULFONATE)
The chemical synthetic routes of sulfonated polyanilines involved
post-treatment of the polymer with fuming sulfuric acid. In the
biochemical synthesis a sulfonated aniline derivative, 2,5
diaminobenzene sulfonate, was polymerized to obtain a water-soluble
polyaniline. The molecular weight of the polymer was on the order
of 18K daltons. The polymerization was also confirmed by FT-IR
spectroscopy. This polymer was soluble at all solution pH
conditions. A plot of absorption spectra of the polymer at
different pH conditions indicated that absorbance at 540 nm
decreased with an increase in solution pH conditions and, at 470
nm, increased with pH. At pH 1.2, the polymer emitted at 380 mn
when excitation was at 320 nm, while at pH 6.0 the emission maximum
shifted to 540 nm. A cyclic voltammogram of the polymer in 0.1M
Tris-HCl buffer at pH 6.0. The peak current followed a linear
relationship with the square root of the scan rate, indicating that
the polymerization was a reversible redox coupling.
POLY(PHENYLAZOANILINE)
Polymerization of phenylazoaniline has been catalyzed by
horseradish peroxidase in the presence of hydrogen peroxide. The
polymer precipitated out of the solution within about half an hour
of reaction initiation, the polymer precipitate was later filtered
off and washed with 50% ethanol to remove unreacted monomers, and
then with water to remove the enzyme. The polymer was soluble in
polar organic solvents like DMF and DMSO with a molecular weight of
about 3000 daltons. The absorption spectra of the polymer and the
monomer of phenylazoaniline in DMF are given in FIG. 13.
The azo groups underwent cis-trans isomerization upon exposure to
light. In this study, we exposed the DMF solution of
poly(phenylazoaniline) to a diffused argon ion laser light for ten
minutes. The polymer was then allowed to relax in ambient
conditions and the absorption spectra were recorded as a function
of time. The difference of the spectra with that before exposure to
laser light is plotted in FIG. 14. It can be observed from the
figure that the polymer underwent conformation changes upon
exposure to the laser light, which relaxed back to a conformation
different than that before excitation. The direction of the arrow
in the figure indicates the decrease in the absorbance. Similar
observation were also made when the polymer was excited with UV
light at 360 nm. This indicated that the polymer had a constrained
structure in solution. The low molecular weight of the polymer
suggested that the backbone, as well as the side chain of the
polymer, underwent cis-trans isomerization resulting in a different
conformation upon relaxation.
POLY(DIAMINOAZOBENZENE)
Poly(diaminoazobenzene) was synthesized from diaminoazobenzene by
oxidative free radical coupling in 20% ethanol solution. The brown
precipitate, which was soluble in polar organic solvents, had a
molecular weight of 80K daltons. The absorption spectrum of the
polymer and the monomer in DMF is presented in FIG. 15.
The cis-trans isomerization of the polymer upon excitation to an
argon ion laser was studied in a situation identical to that of the
poly(phenylene diamine). The different spectra during the course of
relaxation after photoexcitation are provided in FIG. 16. It can be
observed from the figure that the polymer relaxed back to its
original conformation after photoexcitation. The direction of the
arrow in the figure indicates the decrease in the absorbance.
Similar observations were also made upon excitation to UV light at
360 nm. This can be attributed to the higher molecular weight of
the polymer, which allowed only the side chains to undergo
cis-trans isomerization upon photoexcitation. The backbone
isomerization was energetically not favored under these
experimental conditions. It was observed that, upon heating the
solution, where one would expect the backbone to undergo structural
randomization, the polymer underwent irreversible conformation
changes.
POLYPHENOLS AS METAL ION SENSORS
8-hydroxy quinoline is a bidentate ligand, which forms complexes
with metal ions such as Fe(III). 8-hydroxy quinoline-5-sulfonate
(HQS) was enzymatically polymerized to give a water-soluble polymer
(PHQS). PHQS is a polymeric ligand. FIG. 17 shows the carbon-13
spectra of the monomer and the polymer. It was established by
in-situ NMR studies that the oxidative free radical coupling took
place at positions 2, 4 and 7 with the order of preference being
7>2>4.
FIGS. 18 and 19 show the plots of absorbance as a function of
concentration of the metal ion and the ligand for a fixed
concentration of the ligand and the metal ion, respectively. The
lower detection ranges for the metal ion can be achieved by the
proper control of the concentration of the ligand. The ligand has
different complexation capacities with various metal ions. One can
use this polymeric ligand in the immobilized form for a sensitive
metal ion sensor fabrication. With the development of
layer-by-layer multilayer deposition based on the charge neutrality
of polyelectrolytes, these polymeric ligands can be assembled into
organized multilayers that can be used as solid state sensors for
the metal ions.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents of the
invention described specifically herein. Such equivalents are
intended to be encompassed in the scope of the following
claims.
* * * * *